ABSTRACT The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, negative-strand RNA viruses associated with outbreaks of severe viral hemorrhagic fever. The virulence and emerging nature of these zoonotic pathogens makes them a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available. Importantly, there is a major gap in our understanding with regard to the role of host factors at critical stages in the viral replication cycle. The overall goal of this revised R01 application is to characterize EBOV VP30 (eVP30), a key viral protein that facilitates viral transcription, and its interactions with host factors. Our plan builds on recent successes in structurally and functionally characterizing how eVP30 interacts with the viral nucleoprotein (NP) to modulate EBOV RNA synthesis and on a joint (Amarasinghe, Basler, and Krogan groups) unbiased proteomics screen using EBOV proteins as bait that uncovered 193 high-confidence EBOV- human protein-protein interactions (PPIs), including one between eVP30 and the host ubiquitin ligase RBBP6. A crystal structure of this complex revealed that RBBP6 and the viral NP compete for the same VP30 binding surface. Comparison of NP and RBBP6 peptides that bind eVP30 revealed a common PPxPxY motif that is necessary for the interaction. Whereas knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV infectivity, overexpression of RBBP6 or its peptide severely inhibited EBOV transcription and infection. Interestingly, at least two additional eVP30 interactors from our dataset (hnRNP L and hnRNP UL1) also possess PPxPxY motifs. Based on these findings, we propose a multidisciplinary approach to (1) Determine the structure of eVP30 N-terminus and define its association with RNA and protein ligands in the absence and presence of NP; (2) Determine the mechanisms by which eVP30-interacting proteins RBBP6, hnRNP L, and hnRNP UL1 modulate eVP30 function and RNA synthesis; and (3) Test the hypothesis that eVP30 modulates the function of host factors RBBP6, hnRNP L, and hnRNP UL1. These studies will characterize unique host interactions that negatively regulate EBOV replication with the goals of defining how EBOV manipulates host pathways and identifying novel therapeutic targets.